The Nm23 protein has been implicated in a wide variety of biological
processes,including suppression of metastasis, phytochrome responses
inplants, and regulation of differentiation. Here we examine whether
Nm23is involved in Xenopus laevis oocyte maturation. We
found thatNm23 is present in oocytes, indicating that it has the
potentialto be a regulator of maturation. Furthermore, modest
overexpressionof Nm23 inhibited progesterone-induced oocyte
maturation. Thismaturation-inhibitory activity was shared by both the
acidicNm23-H1 isoform and the basic Nm23-H2 isoform and by Nm23
mutantsthat lack nucleoside diphosphate kinase activity (Nm23-H1 H118F
andNm23-H2 H118F). Expression of Nm23 proteins delayed the
accumulationof Mos and the activation of p42 mitogen-activated protein
kinase(MAPK) in progesterone-treated oocytes but had no discernible
effecton Mos-induced p42 MAPK activation. Therefore, Nm23 appearsto
act upstream of the Mos/mitogen-activated protein/extracellular
signal-regulatedkinase kinase/p42 MAPK cascade. These findings suggest
a novelbiological role for Nm23.

Introduction

Nm23 was originally identified as a possible metastasis suppressor
genethrough the discovery that its expression is associated withlow
metastatic potential murine melanoma cell lines (1, 2)
.
Nm23is a member of a gene family that is highly conserved in both
prokaryotesand in eukaryotes. Six human Nm23 family members have been
identified:(a) Nm23-H1 (3)
; (b)
Nm23-H2 (4)
; (c) DR-nm23 (5)
;
(d) Nm23-H4(6)
; (e) Nm23-H5
(7)
; and (f) Nm23-H6 (8)
.

Several lines of evidence suggest that Nm23 has a role in regulating
differentiationand development. Nm23 is a homologue of the
Drosophila awd (abnormalwing disc) gene
(9)
, and mutations in awd are associated witha
variety of developmental defects. During mouse embryonic development,
theaccumulation of Nm23 protein coincides with differentiationof the
embryonic nervous system, heart, and various epithelialtissues during
mouse organogenesis (10)
, again pointing toa possible
role in development. The identification of a differentiation-inhibiting
factor(denoted "I factor") in cell lysates and conditioned media
frommouse myeloid leukemia cells as a member of the Nm23 family
suggestsa role for Nm23 in suppressing differentiation
(11)
. Nm23 expressiondecreases during the differentiation
of hematopoietic cells(12)
, again suggesting a role as an
antidifferentiation factor.In other contexts, Nm23 may promote
differentiation. For example,overexpression of Nm23 promotes neurite
outgrowth in PC12 cells(13, 14)
.

The best-characterized Nm23 proteins exist as homo- or hetero-hexamers
oftwo 152-amino acid isoforms, the acidic Nm23-H1 protein andthe
basic Nm23-H2 protein (15)
. These and other Nm23 proteins
possess
NDP3
kinaseactivity, catalyzing the transfer of the terminal phosphateof
nucleoside triphosphates to NDPs. In some contexts, the NDPkinase
activity of Nm23 appears to be critical for its biologicaleffects. For
example, whereas wild-type Nm23 promotes neuriteoutgrowth, a point
mutant of Nm23 (H118A) that lacks NDP kinaseactivity does not promote
neurite outgrowth and, in fact, suppressesnerve growth factor-induced
neurite outgrowth (13, 14)
.

However, in other contexts, the NDP kinase activity of Nm23appears to
be unimportant. Mutated Nm23 proteins lacking NDPkinase activity still
score as the differentiation-inhibiting"I factor" mentioned
above (16)
. In addition, Nm23-H2 can bindto a
nuclease hypersensitive element on the human c-myc promoter
andtransactivate gene expression in vitro and in
vivo(17, 18)
,and this transactivation is
independent of NDP kinase activity(19)
. Thus, some Nm23
functions appear to be independent ofits NDP kinase activity. Nm23
proteins also function as histidine/asparticacid-specific protein
kinases (20)
and as serine/threonine-specificprotein
kinases (21)
and have been proposed to link receptorsto G
proteins (22)
. However, it is not yet clear how relevant
anyof these activities are for the biological effects of Nm23,nor is
it clear where Nm23 exerts its effects. It may act inthe nucleus (as
suggested by its identification as a myc promoter-binding
protein),in the cytoplasm, or outside the cell (as suggested by its
abilityto inhibit differentiation when applied to cells).

We have been studying the biochemical pathways that regulate
Xenopusoocyte maturation. This process is interesting from
a developmentalperspective because maturation is the final,
rate-limiting stepin the production of a fertilizable egg. The study
of oocytematuration has also yielded important insights into cell
cycleregulation, signal transduction cascades, and translational
control.Fully grown stage VI Xenopus oocytes are naturally
arrestedat the G2-M-phase transition of meiosis
I. Release from theG2-like arrest is stimulated
by progesterone, which initiatesa series of events that culminates in
GVBD (or nuclear envelopebreakdown) and the appearance of a white spot
on animal poleof the oocyte (reviewed in Ref. 23
). Early
after progesteroneexposure, there is a decrease in oocyte cAMP levels
and inhibitionof adenylyl cyclase and protein kinase A. After a lag of
a fewhours, polyadenylation of Mos mRNA occurs, resulting in an
increasein the translation of Mos. Synthesis of Mos leads to the
activationof MEK and p42/extracellular signal-regulated kinase 2 MAPK,
whichfacilitates the activation of maturation-promoting factor, a
proteinkinase composed of Cdc2 and cyclin B. Together, p42 MAPK and
Cdc2/cyclinB trigger the dramatic cell biological changes of oocyte
maturation.

Here we examine whether the ectopic expression of Nm23 proteinscan
facilitate or inhibit oocyte maturation. We found that expressionof
modest concentrations of Nm23-H1 or Nm23-H2 (to 3-fold the
endogenousNm23 level) is sufficient to substantially inhibit
progesterone-inducedoocyte maturation. Catalytically inactive Nm23
proteins alsoinhibited maturation, indicating that this effect does
not dependon the NDP kinase activity of Nm23. Nm23 appears to exert
itseffect upstream of the Mos/MEK/MAPK cascade because it inhibited
progesterone-inducedMos accumulation but did not measurably inhibit
Mos-inducedmaturation. These findings show that Nm23 proteins have the
potentialto be maturation-inhibiting factors.

Results

Oocytes Constitutively Express a Nm23-like Protein.
To determine whether Nm23 might be involved in the early
developmentof Xenopus laevis, we first examined whether
Nm23 proteins arepresent in Xenopus oocytes and eggs. Three
Xenopus Nm23 cDNAs(X1, X2, and X3) have been identified and
shown to be 8287%identical to their human counterparts
(24)
. We used antihumanNm23 antibodies to look for a
cross-reacting Nm23 protein inXenopus oocytes. A putative
Nm23 band with apparent molecularweight of
Mr 24,000 was seen by
immunoblotting (Fig. 1A)
. Itsidentification was supported by its binding to
ATP-Sepharose,a characteristic of Nm23 proteins (25)
.
There was no changein the intensity or apparent molecular weight of
the endogenousNm23 band during progesterone-induced oocyte maturation.

Fig. 1. Effects of Nm23-H1 and Nm23-H1F on maturation. A, Nm23
immunoblot showing the endogenous Mr24,000 Nm23 protein and the ectopic Mr19,000 human Nm23 proteins. Oocytes were microinjected with water
(Lane 1) or synthetic mRNAs for Nm23-H1 (Lanes
24) or Nm23-H1F (Lanes 57). Oocytes were
incubated for 2024 h in the absence of progesterone to allow full
expression of Nm23 and then incubated for 10 h in the presence of
progesterone. BD, time course of
maturation in oocytes microinjected with water (Control,
) or mRNAs for Nm23-H1 () or Nm23-H1F (). The amount of RNA
injected ranged from 974 ng/oocyte, as indicated. GVBD was scored by
the appearance of a white dot at the oocytes animal pole. Ten to 20
oocytes were followed in each experimental group. Data shown are from
one of two similar experiments.

Overexpression of Wild-Type and Catalytically Inactive Mutant Nm23
Delays Progesterone-induced GVBD.
Next we carried out microinjection studies to determine whetherNm23
was a positive or negative regulator of progesterone-inducedoocyte
maturation. Immature oocytes were microinjected withvarious amounts of
wild-type Nm23-H1 or catalytically inactiveNm23-H1F mRNA and incubated
for 24 h to allow the Nm23 proteinsto be expressed. The Nm23 mRNA
did not cause the oocytes tomature and had no obvious effect on oocyte
viability. However,Nm23 expression did have a dramatic effect on the
oocytesresponses to progesterone (5 µg/ml). As shown in Fig.1
,
BD, both Nm23-H1 and Nm23-H1F caused a
dramatic,dose-dependent delay in progesterone-induced maturation. The
delaywas detectable at the lowest doses of RNA used (Fig. 1B)
andincreased with increasing RNA doses (Fig. 1, C and D)
. Thecatalytically inactive Nm23-H1F
protein was more effective,not less effective, than wild-type Nm23-H1
(especially in Fig.1, C and D
). Therefore, the
maturation-delaying effect of Nm23-H1does not depend on the NDP kinase
activity of Nm23.

Fig. 1A
shows the final levels of Nm23-H1 and Nm23-H1F after
34h of expression (24 h in the absence of progesterone followed
by10 h in the presence of progesterone). The lowest amount of
injectedRNA (9 ng/oocyte) resulted in an exogenous Nm23 band about
threetimes the intensity of the endogenous Nm23 band (Fig. 1A)
.Thus, a relatively modest level of Nm23 overexpression
produceda measurable effect on the kinetics of maturation (Fig. 1B)
,suggesting that the endogenous Nm23 protein may also
functionto restrain or oppose maturation. Higher amounts of Nm23 RNA
producedhigher levels of Nm23 protein (Fig. 1A)
and had
more dramaticeffects on the timing of maturation (Fig. 1, C and D)
. Comparableconcentrations (50 ng) of RNA for an
irrelevant protein (catalyticallyinactive Xmkp-1) had no detectable
effect on
maturation,4
arguingthat the inhibition of progesterone-induced maturation
is aNm23-specific effect.

Next we compared the basic Nm23-H2 and Nm23-H2F proteins withthe
acidic Nm23-H1 and Nm23-H1F proteins in terms of their effectson
maturation. Oocytes were microinjected with 50 ng of mRNAfor one
of the four Nm23 proteins. About 24 h after injection,when the
accumulation of Nm23 protein reached maximum levels,half of the
microinjected oocytes were placed in 5 µg/mlprogesterone. As shown
in Fig. 2
, the H2 and H2F forms of Nm23were similar to the H1 and H1F forms in
their ability to delayprogesterone-induced maturation (compare Fig. 2, A and B
).Once again, the catalytically
inactive form (Nm23-H2F) had astronger inhibitory effect than the
active NDP kinase (Nm23-H2;Fig. 2, A and B
).

Fig. 2. The effects of acidic (Nm23-H1 and Nm23-H1F) and basic (Nm23-H2 and
Nm23-H2F) isoforms of Nm23 on progesterone-induced GVBD. Oocytes were
microinjected with mRNA (50 ng/oocyte) for Nm23-H1 (A,
), Nm23-H2 (A, ), Nm23-H1F (B,),
or Nm23-H2F (B, ) or microinjected with water
(A and B, Control,
). Oocytes were incubated for 20 or 24 h for full
expression Nm23 and then stimulated with 5 µg/ml progesterone. At
least 20 microinjected oocytes were followed for each experimental
group, and the results were repeated.

The concentration of progesterone used in the previous experiments(5
µg/ml) is sufficient to induce maximal maturation inmost batches of
oocytes. We also wished to determine the effectof Nm23 expression on
maturation induced by lower concentrationsof progesterone. Oocytes
were injected with 50 ng of Nm23-H1or Nm23-H1F mRNA or with water and
treated with 5, 0.5, 0.1,and 0.01 µg/ml progesterone, in which
concentration 100%of control oocytes could be matured. As shown in
Fig. 3
, theinhibitory effect of the Nm23 mRNAs was more marked when lower
dosesof progesterone were used.

Fig. 3. The effects of Nm23-H1 and Nm23-H1F expression on maturation
induced by various concentrations of progesterone. Oocytes were
microinjected with water (Control, ) or with Nm23-H1
() or Nm23-H1F () mRNA (50 ng/oocyte) and incubated for 2024 h.
The oocytes were then incubated with (A) 5,
(B) 0.5, (C) 0.1, and (D)
0.01 µg/ml progesterone as indicated and scored for GVBD. Data shown
are from one of two similar experiments.

Nm23 Acts Upstream of Mos.
Progesterone causes a rapid, transient decrease in cAMP levelsin
oocytes. This is followed by a lag period of a few hoursthat
culminates in the polyadenylation and translation of theMos message.
Mos, in turn, activates the MAPK cascade proteinsMEK1 and p42 MAPK,
which brings about the activation of Cdc2.We wished to determine
whether Nm23 inhibited maturation bydelaying the synthesis of Mos, by
inhibiting the activationof the MAPK cascade by Mos, or by inhibiting
steps further downstream.

Accordingly, we examined the effect of Nm23 overexpression onthe time
course of Mos accumulation and p42 MAPK phosphorylationin
progesterone-treated oocytes. Oocytes were microinjectedwith water or
with Nm23-H1 and Nm23-H1F mRNAs (50 ng) and incubatedfor 2024 h. As
shown in Fig. 4
, both Nm23-H1 and Nm23-H1Fdelayed the phosphorylation of p42 MAPK
(Fig. 4A)
and the accumulationof Mos (Fig. 4B)
.
These findings suggest that Nm23 exerts itseffects upstream of the
Mos/MEK/p42 MAPK cascade.

Fig. 4. Effects of Nm23-H1 expression on progesterone-induced p42 MAPK
phosphorylation (A) and Mos accumulation
(B) and on Mos-induced p42 MAPK phosphorylation
(C). p42 MAPK (A) and Mos
(B) immunoblots for progesterone-treated oocytes are
shown. Oocytes were microinjected with 50 ng of mRNA encoding Nm23-H1
or Nm23-H1F or with water and incubated for 20 or 24 h. Oocytes
were then incubated with progesterone (0.1 µg/ml) for the lengths of
time indicated. Five oocytes were randomly collected, pooled, and lysed
for each time point. The upper bands in A
represent phosphorylated p42 MAPK. The first two lanes
in A and B are samples of immature
oocytes (-) and mature oocytes (+) run as standards. The
remaining lanes are samples of five oocytes collected at
various times after progesterone treatment and then pooled and lysed.
C, p42 MAPK immunoblots of Mos-injected oocytes. Oocytes
were microinjected with 50 ng of mRNA encoding Nm23-H1 or Nm23-H1F or
with water and incubated for 20 or 24 h. Oocytes were then
microinjected with purified recombinant Mos protein (final
concentration, 180 nM) and incubated for the lengths of
time indicated. Samples of five oocytes were pooled and lysed for each
time point.

In the same experiment, we also examined the effect of Nm23
overexpressionon Mos-induced oocyte maturation. Control oocytes and
Nm23-expressingoocytes were microinjected with purified malE-Mos
protein (afusion protein consisting of maltose-binding protein and
XenopusMos; final concentration, 180
nM), and the time course of p42MAPK
phosphorylation was monitored. As shown in Fig. 4C
, thetime
course of p42 MAPK phosphorylation was similar in all threesets of
oocytes. These findings are again consistent with thehypothesis that
Nm23 exerts its effects on oocyte maturationupstream of the
Mos/MEK/p42 MAPK cascade.

Discussion

Here we demonstrate that human Nm23 can influence the courseof
Xenopus oocyte maturation; overexpression of Nm23-H1 or
Nm23-H2delays progesterone-induced GVBD. The effect depended on the
amountof Nm23 mRNA microinjected and the concentration of progesterone
usedto initiate maturation. Substantial delays were seen under
conditionsin which Nm23 was only modestly overexpressed, suggesting
thatthe endogenous Nm23 protein may also act to oppose or restrain
oocytematuration.

The enzymatically inactive mutants Nm23-H1F and Nm23-H2F werefound to
inhibit progesterone-induced maturation even more effectivelythan the
wild-type proteins. This finding indicates that NDPkinase activity is
not required for Nm23 to negatively regulateprogesterone-induced
oocyte maturation. Kinase-independent effectsof Nm23 have been found
in other contexts as well (16, 19)
.However, in some
contexts, the NDP kinase activity of Nm23 doesappear to be critical
for biological function. For example,neurite outgrowth in PC12 cells
is promoted by Nm23, but notby kinase-inactive forms of Nm23
(14)
. The present resultssuggest that the mutants may
mimic the active rather than inactiveconformation of the wild-type
proteins in some respects. Forexample, the mutants might more readily
undergo serine phosphorylationthan the wild-type proteins. This
hypothesis awaits furtherinvestigation.

Two lines of evidence indicate that Nm23 acts upstream of Mosto
inhibit oocyte maturation: (a) Nm23 expression delays the
accumulationof Mos; and (b) Nm23 does not affect
Mos-induced p42 MAPK activation.Although the signal transduction
pathways upstream of Mos arestill poorly understood, previous work
suggests two points inthe process where Nm23 might exert its
antimaturation effect.Nm23 can interact physically with the
Gs protein (26, 27)and has been
proposed to participate in G protein activation(22)
.
Thus, Nm23 might increase basal Gs activity and
cAMPlevels in the oocyte, accounting for its ability to inhibit
progesterone-inducedmaturation. Nm23 has also been proposed to inhibit
polyadenylationof its own message by binding to sequences similar to
the c-mycnuclease hypersensitive element in its
3'-untranslated region(24)
. Cytoplasmic polyadenylation
of Mos and other mRNAs iscritical for progesterone-induced oocyte
maturation (28, 29,30, 31)
. Thus, Nm23 might inhibit maturation
by interferingwith polyadenylation. Studies are under way in our
laboratoryto test these hypotheses. In addition, the identification of
Nm23-interactingproteins may provide clues as to how Nm23 impinges on
cell signalingin Xenopus oocytes and in other systems as
well.

In Vitro Transcription and Microinjection.
Nm23 mRNA was prepared using an in vitro SP6 transcription
kit(Ambion) and stored in aliquots at -80°C. Oocytes were
microinjectedwith 50 nl of mRNA (50 ng) around the equator of the
oocyte,transferred, and incubated for 1 day to allow Nm23 protein
accumulationto reach maximal levels. Meiotic maturation was induced by
treatingoocytes with 0.15 µg/ml progesterone (Sigma), andGVBD was
assessed after various incubation times. In some experiments,
Nm23-expressingoocytes were subjected to a second microinjection with
malE-Mosprotein (final concentration, 180 nM) to
induce maturation.

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